1a.Objectives (from AD-416):
1. Develop functional genomic resources and employ these resources to examine the genetic basis of biological traits that can potentially be used for biologically based control, including implications for the geographic origins of infestations.

2. Expand current biocontrol efforts by discovering and developing new parasites and pathogens; improving mass culture and field release systems; and defining host specificity of natural enemies.

3. Characterize semiochemicals through investigation of pheromone biosynthesis and release; use these findings to develop novel biologically-based control and surveillance methods, including the detection and treatment of incipient or low level populations. Determine the genetic/chemical bases for differential fire ant sensitivity to corn and turfgrass germplasm and evaluate potential for novel control of fire ants.

4. Develop integrated pest management plans that utilize available control methods, perform comprehensive risk assessment, and that can be adapted to specific stakeholder needs, including local eradication.

1b.Approach (from AD-416):
1. Normalized cDNA libraries will be sequenced using 454-pyrosequencing technology. Resulting sequences assembled and automatically and manually annotated for key words, gene function, and Gene Ontology terms. The 454 data will be used to search for potential microbes infecting fire ants and determine their nature, distribution, relationship, and effects of these microbes in fire ants. SNPs within the data will be identified and applied to high-resolution identification of the source population of introduced S. invicta. Microarrays will be constructed and used to identify differentially expressed genes from parasite-infected and -uninfected fire ants and identify genes co-expressed with the social form-specific gene Gp-9 allelic variants.
2. Microsporidia: After approval, V. invictae will be released in the U.S. following the procedures for K. solenopsae introductions. Phorid Flies: Additional decapitating phorid flies will be released using procedures based on our previous successful releases in the U.S. The impact of phorid flies will be assessed by multiple methods, e.g. monitoring the establishment, expansion, distribution and parasitism rates of phorid fly species across the fire ant range. Viruses: With purified preparations of SINV, a number of basic studies will be conducted, including lethal dose evaluations, colony transmission studies, field-testing, and formulation development.
3. The function of the PBAN/pyrokinin family of neuropeptides will be investigated by: a) in vivo injection into female and male sexuals and immatures and observed for phenotypic change; and b) use of RNAi gene knockout methods ¬ followed by monitoring for phenotypic and/or behavioral changes. Monitoring and surveillance methods will be developed for fire ants, using known attractants. Existing fire ant bioassays will be adapted and applied to non-fire ant invasive pest ant species to create better baits and effective monitoring systems, e.g. attractants and repellents. There are many genetically characterized inbred lines of maize, e.g. yellow and white that will be tested for ant preference/damage at seed germination and seedling stages, and root development differences will be examined (phenotypic traits). Differential gene expression profiles will be used to identify genes of interest. Turfgrasses will be re-examined to determine which species is most inhibitory to fire ant colony development. Long-term molecular studies will identify the genetic basis of observed differences.
4. Potential geographic range expansion of tramp ant species will be modeled using CLIMEX a program that can be used to predict where a pest ant of interest can survive. Phagostimulants will be studied to improve the acceptability of baits for non-Solenopsis pest ants by adapting methods used for fire ants. Standard laboratory colony tests will be used to assess bait formulation effects on brood volume, adult populations, and queen survivorship. A combination of monitoring tools, baits, and biologically based-control methods will be applied to selected invasive ant species as these new tools become available.

3.Progress Report:
Significant progress was made on understanding the unique social biology of ants and in using basic and applied research to develop new ant control methods (biological, insecticide, gene-targeting) and new surveillance tools. Objective 1. Studies were completed to characterize the genomic region responsible for the multiple and single queen forms of social organization in fire ant colonies. This stable, non-combining genomic region is very large and has many of the key properties of sex chromosomes. Importantly, most genes with expression differences between individuals of the two colony social forms reside in this region. Other research found that ants and bees, share a similar, mammalian-like circadian clock. This is the first characterization of clock genes in an ant and is a key step towards understanding socially-regulated flexibility in circadian rhythms. Objective 2. Activities to discover new parasites and pathogens in Argentina were halted until recently by severe restrictions on the export of biological materials. However, a new virus (SiDNV) was discovered in South American fire ants. The virus is not found in U.S. populations making it a candidate for importation and release. Bait formulations (protein-, oil-, and sugar-based) of another fire ant virus (SINV-3) were effective at killing fire ant colonies, supporting its potential use as a biopesticide. In related research, fire ant colonies reared continuously at high temperature (˜86°F) were resistant to the SINV-3 virus. This correlates with the previously determined prevalence of this virus in winter. The decapitating fly, Pseudacteon cultellatus, has survived for another year at two of five sites in Florida. Significant efforts to document the effect of biocontrol releases by monitoring fire ant populations over time in Georgia and Florida revealed that current ant populations are generally lower than in the same sites pre-release of biocontrol agents (1990s). Thus, the biological control agents established in the U.S. are reducing fire ant populations. Objective 3, RNA interference technology continued to be developed as a highly specific control method for fire ants and other pest insects. Experiments with fire ants and moths demonstrated lethal effects for immature and adult life stages, as well as reduced pheromone production. Prototype fire ant specific surveillance devices were developed and evaluated that photographed attracted ants and transmitted the photos via internet to a computer for remote ant identification. Concomitantly, methods to rapidly identify fire ants continue to be developed. These technologies will provide regulatory agencies (e.g. APHIS, EPA, Homeland Security) with needed surveillance and identification tools. Objective 4. Studies to develop integrated pest management strategies for the invasive tawny crazy ant indicated that liquid ant bait was widely dispersed from a prototype bait station. This strategy has the potential to extend control into inaccessible areas that serve as reservoirs for reinvasion.

4.Accomplishments
1.
Potential biological control agent discovered in South American fire ants. Natural enemies for fire ant control are largely absent in U.S. populations of fire ant resulting in unchecked population growth and expansion. ARS researchers at Gainesville, Florida, have discovered a virus in South American populations of fire ants with potential to provide sustainable control of this invasive ant pest in the U.S. This discovery represents the first opportunity for virus-based classical biological control of fire ants in the U.S.

2.
Virus-containing baits found effective against fire ants in the laboratory. Natural alternatives to traditional insecticides are not available for fire ant control. ARS researchers at Gainesville, Florida, have determined that fire ant-specific viruses can control fire ant colonies when incorporated into baits and fed to the colonies. Additional studies are underway to improve formulations and increase impacts on fire ant colonies. The discovery represents the first virus-based biopesticide for fire ant control.

3.
Gene expression patterns identified for founding fire ant queen behavioral phenotypes. Little is known about the effect of social environment on gene expression during the crucial newly mated queen colony foundation stage. ARS researchers at Gainesville, Florida, determined that social environment played a major role in the determination of the patterns of gene expression, while the physiological state and the social rank of founding queens are only secondary. These results highlight the powerful influence of social environment on regulation of gene expression patterns, physiology and, ultimately, social behavior.

4.
The genomic region responsible for single and multiple queen social organizational forms in fire ants identified. Introduced fire ants exhibit two colony social forms that differ in the number of reproductive queens per colony as well as a host of other traits. Remarkably, many individual and life-history traits allied to social organization also are associated with allelic variation at the single gene Gp-9. ARS researchers at Gainesville, Florida, found Gp-9 is embedded in an epistatic network of genes that are resistant to being broken up and that regulate various aspects of the complex social syndrome (i.e., a “supergene”). The newly discovered non-recombining genomic region (part of a pair of heteromorphic chromosomes) has many of the key properties of sex chromosomes and comprises most of the genes with demonstrated expression differences between individuals of the two social forms. These findings highlight how genomic rearrangements can maintain divergent adaptive social phenotypes involving many genes acting in concert by locally limiting recombination.

5.
Multiple uses of fire ant alarm pheromone. The alarm pheromone in ants is usually associated with the rapid movement of workers after disturbance, but female and male sexuals (winged forms) also produce the alarm pheromone, suggesting additional functions. ARS researchers at Gainesville, Florida, determined that male and female sexuals initiate mating flights with the alarm pheromone and that males exhaust their pheromone supply while maintaining a lek into which the females fly for mating. In contrast, newly mated queens accumulate large amounts during colony growth that dissipates to insignificant amounts in mature colonies. Results support a diverse multifunctional role for the alarm pheromone in fire ants.

6.
New low-cost diet for rearing fire ants. Laboratory studies of fire ants require healthy growing colonies. ARS researchers at Gainesville, Florida, discovered that fire ant colonies can be successfully reared on raw beef liver and sugar water. This diet is not as good as domestic crickets, but it is about 1/4 the cost, easily available, more effective than any artificial diet, better than mealworms and other cricket species, and sufficient for rearing and maintaining stock colonies. A liver and sugar water diet may also prove very useful to researchers around the world because this diet is also effective at rearing many other kinds of ants.

7.
Insect growth regulator reduces invasive crazy ant colony growth. The tawny crazy ant is an invasive pest that is spreading in the southern U.S. Ant baits are an efficient and environmentally compatible method of control, but most commercial baits are ineffective against this species. ARS researchers at Gainesville, Florida, demonstrated that bait containing an insect growth regulator significantly reduced colony growth of the tawny crazy ant. An effective insect growth regulating bait has the potential to be more effective than standard bait toxicants because it can be more easily distributed by the ants among their numerous colonies.